灰树花多糖对EV71病毒抑制作用的研究

发布时间：2018-11-15 06:40

【摘要】：目的：手足口病是一种常见的传染疾病,以婴幼儿发病多见,肠道病毒71型(Enterovirus71, EV71),属于小核糖核酸病毒家族中的肠道病毒属成员,是引起手足口病的常见病因,到目前为止,临床上还没有十分有效的治疗方案或者疫苗研制成功。在中国近几年的数次手足口病爆发的过程中,中草药显示出了良好的临床治疗效果,灰树花多糖被发现具有多种抗癌,抗炎,抗病毒等药用价值,然而,在灰树花多糖抗EV71病毒的研究方面,尚未见报道。因此,我们探讨了灰树花多糖的抗EV71病毒活性,同时对多糖的结构进行了分析,以期为进一步了解这一生物活性物质的构效关系提供一定的基础。方法：实验中采用傅里叶红外光谱仪和核磁共振仪(包括核磁共振氢谱仪和核磁共振碳谱仪)被用于研究纯化后的灰树花多糖的结构,排除测定药物对细胞增值的影响后,我们初步用结晶紫染色法检测药物的抗EV71病毒作用,采用蛋白印迹法和实时定量PCR进一步说明了灰树花多糖的抗病毒作用及其作用的初步机制,通过时间进程实验我们也进一步研究了灰树花多糖抗病毒的时间点。结果：通过体外实验发现灰树花多糖可以通过抑制病毒前体蛋白合成和RNA合成来阻碍EV71的复制,在病毒感染的早期,灰树花多糖可以有效地抑制EV71的作用,通过对VP1蛋白水平检测及caspase-3蛋白的检测,证明灰树花多糖通过抑制EV71诱导的caspase-3的剪切活性,从而起到抑制宿主细胞凋亡的作用。EV71感染可以引起细胞内IκBα的下调,灰树花多糖可以通过抑制这种由EV71感染诱导的宿主细胞IκBα蛋白下调的表达达到抗病毒的作用。结论：实验中所用灰树花多糖含有α—吡喃型糖残基和β—葡萄糖残基糖苷键,具有良好的抑制EV71病毒复制的作用。
[Abstract]:Objective: hand, foot and mouth disease is a common infectious disease. Enterovirus 71 (Enterovirus71, EV71), a member of small RNA virus family, is a common cause of hand, foot and mouth disease. So far, there is no very effective treatment or vaccine development. In the course of several outbreaks of hand, foot and mouth disease in China in recent years, Chinese herbal medicine has shown a good clinical therapeutic effect. Polysaccharides from Grifola frondosa have been found to have many medicinal values such as anticancer, anti-inflammatory, anti-virus, etc. However, The study of Grifola frondosa polysaccharides against EV71 virus has not been reported. Therefore, we discussed the antiviral activity of Grifola frondosa polysaccharides and analyzed the structure of polysaccharides in order to provide a basis for further understanding the structure-activity relationship of this bioactive substance. Methods: Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) were used to study the structure of purified Grifola frondosa polysaccharides. The antiviral effect of the drug against EV71 was detected by crystal violet staining. The antiviral effect of Grifola frondosa polysaccharides and its mechanism were further explained by Western blotting and real-time quantitative PCR. We also studied the antiviral time point of Grifola frondosa polysaccharide by time process experiment. Results: it was found in vitro that Grifola frondosa polysaccharides could inhibit the replication of EV71 by inhibiting the synthesis of virus precursor protein and RNA. In the early stage of virus infection, Grifola frondosa polysaccharides could effectively inhibit the effect of EV71. By detecting the level of VP1 protein and caspase-3 protein, it was proved that Grifola frondosa polysaccharide inhibited the apoptosis of host cells by inhibiting the shearing activity of caspase-3 induced by EV71. EV71 infection could induce the down-regulation of I 魏 B 伪 in cells. Grifola frondosa polysaccharides can inhibit the down-regulation of I 魏 B 伪 protein in host cells induced by EV71 infection. Conclusion: the polysaccharides of Grifola frondosa contained 伪 -pyrrolidene glycosyl residues and 尾 -glucose residues glycosides, which could inhibit the replication of EV71 virus.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R725.1

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